DESCRIPTION: The general aim of the proposed research is to determine the high resolution structures of membrane proteins and protein complexes in bilayer environments. The research takes advantage of magic angle spinning NMR methods for measuring internuclear distances between specific sites in proteins. Accurate 13C...13C and 13C...15N distance measurements in the range of 3-6 A make it possible to establish the local secondary structure and tertiary interactions between polypeptide chains spanning membrane bilayers. The research has three objectives. The first objective is to calibrate the distances derived from several NMR approaches using small crystals of a peptide, leu-enkephalin, whose structure has previously been determined to atomic resolution by diffraction methods. These studies are designed to establish the accuracy and resolution of distance measurement using RR (rotational resonance) and RFDR (rf-driven recoupling) NMR pulse sequences. The second objective is to determine the structure of the transmembrane domain dimer of glycophorin A. Noncovalent interactions between transmembrane helices appear to be important in driving protein oligomerization and there are likely to be different motifs that direct specific helix association. The studies on glycophorin A are the first to map out the key interactions for a dimerization motif. The third objective is to determine the three-dimensional structure of phopholamban, a 52-residue protein that forms a pentameric complex in membranes. This will be the first high resolution structure determined in a membrane bilayer environment of a protein complex having multiple membrane-spanning domains. Structural studies on both phosphorylated and non-phosphorylated forms of the protein will address the mechanism by which the protein regulates calcium levels across cardiac cell membranes. The proposed research forms the basis for understanding the molecular mechanism of signal transduction across cell membranes mediated by protein dimerization and ion conductance by a stable pore forming protein complex.